Stimulation of yeast telomerase activity by the ever shorter telomere 3 (Est3) subunit is dependent on direct interaction with the catalytic protein Est2.

Telomerase is a multisubunit enzyme that maintains genome stability through its role in telomere replication. Although the Est3 protein is long recognized as an essential telomerase component, how it associates with and functions in the telomerase complex has remained enigmatic. Here we provide the first evidence of a direct interaction between Saccharomyces cerevisiae Est3p and the catalytic protein subunit (Est2p) by demonstrating that recombinant Est3p binds the purified telomerase essential N-terminal (TEN) domain of Est2p in vitro. Mutations in a small cluster of amino acids predicted to lie on the surface of Est3p disrupt this interaction with Est2p, reduce assembly of Est3p with telomerase in vivo, and cause telomere shortening and senescence. We also show that recombinant Est3p stimulates telomerase activity above basal levels in vitro in a manner dependent on the Est2p TEN domain interaction. Together, these results define a direct binding interaction between Est3p and Est2p and reconcile the effect of S. cerevisiae Est3p with previous experiments showing that Est3p homologs in related yeast species influence telomerase activity. Additionally, it contributes functional support to the idea that Est3p is structurally related to the mammalian shelterin protein, TPP1, which also influences telomerase activity through interaction with the Est2p homolog, TERT.

Structural bases of dimerization of yeast telomere protein Cdc13 and its interaction with the catalytic subunit of DNA polymerase α.

Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Here, we report the crystal structure of the N-terminal OB (oligonucleotide/oligosaccharide binding) fold of Cdc13. Although Cdc13 has an RPA70-like domain organization, the structures of Cdc13 OB folds are significantly different from their counterparts in RPA70, suggesting that they have distinct evolutionary origins. Furthermore, our structural and biochemical analyses revealed unexpected dimerization by the N-terminal OB fold and showed that homodimerization is probably a conserved feature of all Cdc13 proteins. We also uncovered the structural basis of the interaction between the Cdc13 N-terminal OB fold and the catalytic subunit of DNA polymerase α (Pol1), and demonstrated a role for Cdc13 dimerization in Pol1 binding. Analysis of the phenotypes of mutants defective in Cdc13 dimerization and Cdc13-Pol1 interaction revealed multiple mechanisms by which dimerization regulates telomere lengths in vivo. Collectively, our findings provide novel insights into the mechanisms and evolution of Cdc13.

Is there a telomere-bound 'EST' telomerase holoenzyme?

Eukaryotic linear chromosomes culminate in nucleoprotein structures designated telomeres. The terminal telomeric DNA consists of tandem repeats of a G-rich motif that is established and maintained by the action of the specialized reverse transcriptase telomerase. In addition to the core enzyme, effective replication of telomeric DNA requires a number of regulatory factors. In budding yeast, four components identified in the seminal Ever Shorter Telomere (EST) genetic screens constitute the keystone proteins that sustain telomeric DNA. Importantly, the EST proteins appear to be structurally conserved from yeast to human. The mechanism of telomerase recruitment and regulation, with an emphasis on the EST proteins, are discussed in this review.

HSP90 manages the ends.

The telomere environment requires an efficient means to assemble and disassemble a multitude of structures to operate correctly and to help achieve cellular homeostasis. Telomeres are challenged by a common binding specificity displayed by many of the protein components for telomeric DNA, which could result in competitive DNA interactions, and by a cell cycle-restricted timing of events, which enforces a narrow working period in which to perform numerous tasks. In this review, we discuss how the HSP90 molecular chaperone network avoids these obstacles and facilitates an effective operation of the telomere system.

Slowing bacterial translation speed enhances eukaryotic protein folding efficiency.

The mechanisms for de novo protein folding differ significantly between bacteria and eukaryotes, as evidenced by the often observed poor yields of native eukaryotic proteins upon recombinant production in bacterial systems. Polypeptide synthesis rates are faster in bacteria than in eukaryotes, but the effects of general variations in translation rates on protein folding efficiency have remained largely unexplored. By employing Escherichia coli cells with mutant ribosomes whose translation speed can be modulated, we show here that reducing polypeptide elongation rates leads to enhanced folding of diverse proteins of eukaryotic origin. These results suggest that in eukaryotes, protein folding necessitates slow translation rates. In contrast, folding in bacteria appears to be uncoupled from protein synthesis, explaining our findings that a generalized reduction in translation speed does not adversely impact the folding of the endogenous bacterial proteome. Utilization of this strategy has allowed the production of a native eukaryotic multidomain protein that has been previously unattainable in bacterial systems and may constitute a general alternative to the production of aggregation-prone recombinant proteins.

The conserved Est1 protein stimulates telomerase DNA extension activity.

The first telomerase cofactor identified was the budding yeast protein Est1, which is conserved through humans. While it is evident that Est1 is required for telomere DNA maintenance, understanding its mechanistic contributions to telomerase regulation has been limited. In vitro, the primary effect of Est1 is to activate telomerase-mediated DNA extension. Although Est1 displayed specific DNA and RNA binding, neither activity contributed significantly to telomerase stimulation. Rather Est1 mediated telomerase upregulation through direct contacts with the reverse transcriptase subunit. In addition to intrinsic Est1 functions, we found that Est1 cooperatively activated telomerase in conjunction with Cdc13 and that the combinatorial effect was dependent upon a known salt-bridge interaction between Est1 (K444) and Cdc13 (E252). Our studies provide insights into the molecular events used to control the enzymatic activity of the telomerase holoenzyme.

The Hsp82 molecular chaperone promotes a switch between unextendable and extendable telomere states.

Distinct protein assemblies are nucleated at telomeric DNA to both guard the ends from damage and lengthen the DNA after replication. In yeast, Cdc13 recruits either Stn1-Ten1 to form a protective cap or the telomerase holoenzyme to extend the DNA. We have established an in vitro yeast telomere system in which Stn1-Ten1-unextendable or telomerase-extendable states can be observed. Both assemblies are Cdc13 dependent, as the Cdc13 C-terminal region supports Stn1-Ten1 interactions and the N-terminal region contains a telomerase-activation function. Notably, the yeast Hsp90 chaperone Hsp82 mediates the switch between the telomere capping and extending structures by modulating the DNA binding activity of Cdc13. Taken together, our data show that the Hsp82 chaperone facilitates telomere DNA maintenance by promoting transitions between two operative complexes and by reducing the potential for binding events that would otherwise block the assembly of downstream structures.

HSP90: the Rosetta stone for cellular protein dynamics?

The Hsp90 proteomic network is expansive and includes a variety of cell processes operating within the cytoplasm and nucleoplasm. Though the functional significance of the extensive interactions has not been defined, we suggest that the Hsp90 molecular chaperone machinery promotes dynamic behaviors for client proteins that is critical to achieve homeostasis. A general rapid action by cell factors would permit both proper assembly of biological complexes and efficient transitions between distinct structures. Here, we describe why the properties that are inherent to molecular chaperones place these proteins in a unique position to drive the dynamic cellular environment.

The hsp90 molecular chaperone modulates multiple telomerase activities.

The Hsp90 molecular chaperone is a highly abundant eukaryotic molecular chaperone. While it is understood that Hsp90 modulates a significant number of proteins, the mechanistic contributions made by Hsp90 to a client protein typically are not well understood. Here we investigate the yeast Hsp90 regulatory roles with telomerase. Telomerase lengthens chromosome termini by specifically associating with single-stranded telomeric DNA and appending nucleotides by reverse transcription. We have found that the yeast Hsp90 homolog Hsp82p promotes both telomerase DNA binding and nucleotide addition properties. By isolating telomerase from different allelic backgrounds we observed distinct defects. For example, in an hsp82 T101I strain telomerase displayed decreased nucleotide processivity, whereas both DNA binding and extension activities were lowered in a G170D background. The decline in telomerase DNA binding correlated with a loss of Hsp82p association. No matter the defect, telomerase activity was recovered upon Hsp82p addition. Importantly, telomere length and telomerase telomere occupancy was yeast Hsp90 dependent. Taken together, our results indicate that Hsp82p promotes telomerase DNA association and facilitates DNA extension once telomerase is engaged with the DNA.

Purification and characterization of enterocin 62-6, a two-peptide bacteriocin produced by a vaginal strain of Enterococcus faecium: Potential significance in bacterial vaginosis.

A bacteriocin produced by a vaginal isolate of Enterococcus faecium strain 62-6, designated enterocin 62-6, was characterized following purification and DNA sequence analysis and compared to previously described bacteriocins. Enterocin 62-6 was isolated from brain heart infusion (BHI) culture supernatants using ammonium sulfate precipitation followed by elution from a Sepharose cation exchange column using a continuous salt gradient (0.1-0.7 M NaCl). SDS-PAGE of an active column fraction resulted in an electrophoretically pure protein, which corresponded to the growth inhibition of the sensitive Lactobacillus indicator strain in the gel overlay assay. Purified enterocin 62-6 was shown to be heat- and pH-stable, and sensitive to the proteolytic enzymes alpha-chymotrypsin and pepsin. Results from mass spectrometry suggested that it comprised two peptides of 5206 and 5219+/-1 Da, which was confirmed by DNA sequence analysis. The characteristics of enterocin 62-6 as a small, heat- and pH-stable, cationic, hydrophobic, two-peptide, plasmid-borne bacteriocin, with an inhibitory spectrum against a broad range of Gram-positive but not Gram-negative bacteria, were consistent with its classification as a class IIc bacteriocin. Furthermore, its wide spectrum of growth inhibitory activity against Gram-positive bacteria of vaginal origin including lactobacilli, and stability under the acidic conditions of the vagina, are consistent with our hypothesis that it could have potential significance in disrupting the ecology of the vaginal tract and pave the way for the establishment of the abnormal microbiota associated with the vaginal syndrome bacterial vaginosis. This is the first class IIc bacteriocin produced by a strain of E. faecium of vaginal origin to be characterized.